From the beginning of agriculture humans have been confronted with the problem of plant disease. Throughout history many strides have been made against plant diseases as exemplified through the use of hybrid plants, pesticides and improved agricultural practices. However, as any farmer, home gardener, or houseplant devotee can attest, the problems of plant disease are an ongoing and constant problem in plant cultivation. This invention constitutes a major step forward in solving the problem of plant disease by exploiting a long known fact regarding the method by which certain microbes, especially fungi, attack plants. Specifically, this invention addresses a means by which the etiologic (disease causing) agent is slowed or prevented from actually entering into the plant tissue, thereby preventing disease. Alternatively, in instances where complete invasion is not prevented the present invention confers upon plants a means for reducing the effects of infection thereby reducing or preventing plant mortality due to the disease.
In order for a plant pathogen to infect a plant, it must be able to gain access into and subsequently throughout the plant. Plant pathogens accomplish this in various ways. Generally, this is accomplished by the secretion of chemical substances that affect certain component and/or metabolic mechanisms of the plant to be attacked, i.e. the host for the pathogen. The main groups of compounds that are secreted by plant pathogens and that are related to the disease-causing mechanism are toxins, enzymes, polysaccharides and/or other effectors of growth. One such chemical compound is oxalic acid or oxalate which can be degraded by the enzyme oxalate oxidase. Although the reaction catalyzed by oxalate oxidase is well-known, the physico-chemical attributes of the enzyme as exemplified by barley oxalate oxidase are incorrectly reported in the literature. The secretion of oxalic acid as a means by which plant pathogens attack plant hosts is commonly found in a plurality of fungal genera and especially in the genera Sclerotinia, Sclerotium, Aspergillus, Streptomyces, Penicillium, Pythium, Paxillus, Mycena, Leucostoma, Rhizoctonia and Schizophyllum. These genera of fungi, especially those fungi of the genus Sclerotinia, are known to cause destructive and fatal diseases of numerous highly cultivated plants including field crops such as sunflower, soybean, beans in general, rape/canola, alfalfa, flax, safflower, peanut and clover, vegetable crops such as lettuce, tomato, cucurbits, potato, carrot, radish, pea, lentils, cabbage, broccoli and brussel sprouts, flowers such as petunia and pyrethrum and tree species such as peach. The diseases include not only pre-harvest diseases in the field but also post-harvest diseases during shipping and storage.
The symptoms caused by the aforementioned fungal genera vary with the host plant and the parts of the host plant infected with the disease, as well as being dependant upon environmental conditions at the time of pathogen attack. For example, a feature of all Sclerotinia disease is wilting and collapse of the leaves whereupon the fungus rapidly invades the "heart" of the plant and throughout the stem. The disease is fatal.
In liquid cultures of Sclerotinia levels of oxalic acid ranging from a thousand to ten thousand parts per million, depending upon growth media, age of the culture and other parameters have been observed. Certain plant tissue such as leaves of bean and sunflower exposed to low concentrations of oxalic acid readily show signs of wilting and cell death suggesting the importance of oxalic acid in later stages of disease. The precise mechanism of the disease causing function of oxalic acid after infection is unknown although theories range from chelation of divalent metals interfering with plant cell walls and/or key metabolic enzymes to providing an optimum micro-environment for the action of hydrolytic enzymes secreted by these fungi. Regardless, it is clear from extant evidence that oxalic acid is an integral component of pathogenic attack. Evidence for this conclusion has been obtained by several studies including investigations with oxalate-minus Sclerotinia mutants that appear to possess the normal complement of hydrolytic enzymes and other factors but do not produce disease symtomology. Revertants of these oxalate minus mutants demonstrated normal disease development and characteristics.
The high degree of virulence of diseases associated with the aforementioned fungal genera are well-known. For example, leaves of greenhouse grown sunflower plants infected with Sclerotinia sclerotiorum frequently exhibit wilting and interveinal necrosis three to five days after inoculation. Study of these plants has shown a wilt inducing substance in water extracts of hypocotyl (the part of the axis of a plant embryo or seedling below the cotyledon) lesions. Chemical tests including thin layer chromatography and gas-liquid chromatography have demonstrated that this wilt inducing substance contained oxalic acid and that wilted leaves from infected plants contained over fifteen times more oxalic acid than leaves of healthy plants. As already noted, the oxalic acid moves systemically through the plant to cause disease symptoms in tissues that are both distant from the initial point of infection and not necessarily infected with fungal hyphae.
With this backdrop, the inventors realized that appropriate identification, isolation and expression of an oxalate degrading enzyme such as an oxalate oxidase, oxalate decarboxylase or similar enzyme by a plant might well diminish the pathogenicity of fungi which secrete oxalic acid as a key component of pathogenicity. Accordingly, the inventors set out and achieved the goal of properly identifying and isolating a gene for a protein that is suitable for the introduction of oxalate oxidase activity into plants and microbes using the techniques of genetic engineering.
In particular, the inventors have characterized an enzyme useful in thwarting pathogenicity involving oxalic acid. This uniquely characterized oxalate oxidase enzyme catalyzes or otherwise contributes to a reaction involving the oxidative degradation of oxalate to produce carbon dioxide and hydrogen peroxide. The general form of this reaction is: oxalate+O.sub.2 oxalate/oxidase.fwdarw.2CO.sub.2 +H.sub.2 O.sub.2. The study of this enzyme has resulted not only in its identification, isolation and expression, but also in its characterization and its cloning so that a gene for expression of the enzyme is extant, can be introduced into plants, and expressed by plants thereby conferring disease resistance to fungi in which oxalic acid is a critical component. Such plant transformation would protect the transformed plants against the deleterious disease causing effects of oxalic acid.
It will be appreciated that the applications of the aforementioned inventions are not limited to plant pathogenesis. Still another benefit of this invention is the introduction of an oxalate oxidase gene into a plant to produce a low oxalic acid plant. This could be especially beneficial in high oxalate plants such as peanuts, beets, spinach, rhubarb, barley, cocoa, and many grasses. See, Libert and Franceschi (1987), J. Agric. Food Chem., 35:926-938. Also, the invention has application for the large scale production of oxalate degrading enzymes. The need for such large scale oxalate degrading enzymes is known in a variety of fields including the need for their use in assay kits to determine the presence and/or amount of oxalic acid and for use in the degradation of oxalic acid present in foodstuffs, beverages and commercial processes. For example, a microbial oxalate decarboxylase has been used by the brewing industry (U.S. Pat. No. 4,652,452) and as set forth herein oxalate can also be degraded using oxalate oxidase.
It is, therefore, an object of this invention to fully purify and properly characterize an oxalate oxidase.
It is another object of this invention to isolate, characterize and construct a gene which can express oxalate oxidase in microbes and plants.
It is a further object of this invention to introduce an oxalate oxidase expressing gene into plants thereby conferring on such plants resistance to diseases, especially fungal diseases in which oxalic acid is a major component such as in diseases of the fungal genera Sclerotinia, Sclerotium, Aspergillus, Streptomyces, Penicillium, Pythium, Paxillus, Mycena, Leucostoma, Rhizoctonia and Schizophyllum.